Establishing an organ culture model to study scleral collagen remodeling in myopia.

摘要

Purpose: Increasing evidence suggests that unknown scleral remodeling mechanisms underlie myopia development. We are proposing a novel organ culture system in combination with two-photon fluorescence imaging to quantity collagen remodeling at the tissue- (macro-level) and lamellar-level (micro-level).;Methods: Tree shrew scleral shells were cultured up to 7 days in serum-free media and cellular viability was investigated under: (i) minimal tissue manipulations; (ii) removal of intraocular tissues; (iii) gluing the eye to a stainless steel washer with 50 microL of glue; (iv) same as condition (iii) with 200 microL of glue; (v) supplementing media with Ham's F-12 Nutrient Mixture; and (vi) culturing eyes subjected to 15 mmHg intraocular pressure in a bioreactor. Two different bioreactors were preliminarily tested before a design was finalized. Using our finalized bioreactor, we cultured and fluorescently labeled two scleral shells of normal juvenile tree shrews. Using two-photon microscopy, grid patterns were photobleached into multiple collagen lamellae and across the apex of the scleral shell. These patterns were imaged daily for 3 days, and tissue-/lamella-level strains were calculated from the deformed patterns.;Results: No significant reduction in cell viability was observed under condition (i). All tissue manipulations lowered the average viability. Compared to condition (i), cell viability was significantly reduced at day 0 in condition (ii), at day 3 in conditions (ii, iii, iv, vi), and at day 7 in conditions (ii, iii, iv). One bioreactor was determined superior due to its closed design and ability to be used with a multiphoton microscope. Micro- and macro-level normal strains were -0.36+/-1.51% and 1.22+/-0.74% at day 2 to -0.19+/-1.90% and 2.31+/-1.41% at day 3, respectively. Intralamellar shear angle was 0.63º (IQR = 0.34--1.07) on day 2, and 0.62º (IQR = 0.26--1.12) on day 3.;Conclusions: Findings suggest that tree shrew sclera can be cultured in serum-free media for 7 days with no significant reduction in cell viability. Scleral fibroblasts are highly sensitive to mechanical tissue manipulations and tissue gluing, while Ham's F-12 Nutrient Mixture has a protective effect on cell viability. This is the first study to quantify collagen remodeling deformations over a prolonged period in organ culture suggesting that scleral remodeling in juvenile tree shrews involves tissue elongation, lamellar elongation, and intralamellar deformations.;Keywords: myopia, organ culture, bioreactor, biomechanics, collagen remodeling.